A cultured rat mast cell line, the RBL-2H3 cell, was shown to produce the cytokine, TNFalpha, at physiologically active concentrations, in response to stimulation with antigen, or the combination of Ca2+-ionophore and phorbol myristate and in cells transfected with the gene for the muscarinic m1 receptor by carbachol. Production and release of TNFalpha were two distinct processes although both were dependent on continued stimulation of the cell. Production of TNFalpha was stimulated by an elevation in cytosolic Ca2+ by or the activation of protein kinase C. A strong synergizing signal, possibly the activation of tyrosine kinase, was apparent in antigen-stimulated cells. All other stimulants were weak promoters of TNFalpha production compared to antigen although all stimulants were capable of producing maximal release of secretory granules. There was no evidence that TNAalpha was incorporated into secretory granules, although the pathway for TNFalpha-release was apparently regulated by protein kinase C and Ca2+. Inhibitors of protein kinase C and EGTA blocked release of TNFalpha. Also, because the kinetics of release was the same for all stimulants, in contrast to production of TNFalpha, release was probably dependent on protein kinase C and Ca2+ alone. Studies with dexamethasone revealed other potential transducers for stimulatory signals for production of TNFalpha, namely the GTP binding protein Galphaz and the proto oncogene products of cfos cjun. For example, over-expression of Galphaz in transfected RBL-2H3 cells resulted in markedly enhanced production of TNFalpha and treatment with dexamethasone, which normally decreased expression of Galphaz and TNFalpha production, was less effective in reducing antigen-induced TNFalpha production in the transfected cells. Transfection also altered expression of mRNA for the efos/cjun proto oncogene products after antigen stimulation.